ChIP-seq Analysis of Kidney Injury

Chip assays were performed as described previously^{58 (link)}. Briefly, snap-frozen kidney cortex from SHAM or IRI mice (day 2) were ground into powder in liquid nitrogen. After 15 min chromatin cross-linking was carried out in 1% formaldehyde at room temperature and the reaction was quenched with 0.125 M glycine. After cell lysis and 22 min of sonication, sheared chromatin was incubated with 10 µg of following antibodies: anti-H3K27ac (Abcam, ab4729), anti-H3K4me3 (Millipore, 17-614), anti-BRD4 (Bethyl Laboratory, A301-985A), anti-RNA Polymerase II (Abcam, ab5408), anti-BRD2 (Bethyl Laboratory, A301-583A), anti-BRD3 (Bethyl Laboratory, A301-368A), anti-HNF4A (Abcam, ab41898), anti-GR (Thermo Scientific, PA1-511A), anti-STAT3 (Santa Cruz, sc-482) and anti-STAT5 (Santa Cruz, sc-835). Libraries for next-generation sequencing were prepared with NEBNext Ultra II DNA Library Prep Kit (New England BioLabs, E7645) and sequenced on HiSeq 2500 machine (Illumina).

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Wilflingseder J., Willi M., Lee H.K., Olauson H., Jankowski J., Ichimura T., Erben R., Valerius M.T., Hennighausen L, & Bonventre J.V. (2020). Enhancer and super-enhancer dynamics in repair after ischemic acute kidney injury. Nature Communications, 11, 3383.

Publication 2020

anti-H3K27ac anti-H3K4me3 anti-RNA Polymerase II anti-HNF4A anti-GR anti-STAT3 anti-STAT5 NEBNext Ultra II DNA Library Prep Kit HiSeq 2500 machine

A301 Antibodies Brd4 Cell Chip assays Chromatin Dna library Formaldehyde Frozen Glycine H3k4me3 Kidney cortex Mice Nitrogen Powder Rna polymerase ii Stat3 Stat5

Corresponding Organization :

Other organizations : Brigham and Women's Hospital, Harvard University, National Institutes of Health, University of Veterinary Medicine Vienna

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Variable analysis

independent variables

Mice (SHAM or IRI)

dependent variables

Chromatin modifications (H3K27ac, H3K4me3)
Transcription-related proteins (BRD4, RNA Polymerase II, BRD2, BRD3, HNF4A, GR, STAT3, STAT5)

control variables

Chromatin preparation (snap-frozen kidney cortex, ground into powder in liquid nitrogen, cross-linking, cell lysis, sonication)
Chromatin immunoprecipitation (10 µg of antibodies)
Library preparation (NEBNext Ultra II DNA Library Prep Kit)
Sequencing (HiSeq 2500 machine)

controls

Positive control: Not specified.
Negative control: Not specified.

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